Project Summary: Microelectrode arrays provide a unique, new method for monitoring binding events between small molecules and a biological target in ?real-time?. The method is inexpensive, easy to do using commercially available equipment, and does not require the labeling of either the molecules or the biological target being studied. In addition, the use of a microelectrode array combines that analytical capability with powerful synthetic capabilities that allow for the controlled construction and characterization of spatially addressable molecular libraries. The result is a unique opportunity to expand the utility of surface-based, ?real-time? signaling methods to include experiments that are otherwise impossible. With this said, there are still significant challenges that remain. The synthesis of libraries on an array still involves the placement of individual members of the library on the array by the electrodes using a limited number of reactions. This limits the size of a library that can be made. The analytical techniques on the arrays frequently amplify signals so there is a need to calibrate the arrays by controlling the concentration of ligands on the surface. How can this be accomplished without sacrificing the ability to characterize and tune the surface of the array. Finally, it is one thing to state that new synthetic capabilities afford new analytical opportunities, but what are these opportunities. It is the goal of the propose work to address these issues by pursuing three main objectives. First, new site-selective methods for parallel synthesis will be developed so that molecular scaffolds that are either placed or built on an array can be diversified directly on that array. In this way, larger libraries can be synthesized directly onto the arrays thus avoiding the time and expense of transferring them to an array one member at a time. Second, the methodology needed to generate concentration gradients of a ligand on the arrays will be developed. This will allow calibration of the arrays so that he data gathered can be compared with alternative methods and solution-phase data and analyzed for potential avidity events. Third, analytical experiments on the arrays will be combined with FRET studies to illustrate how the binding and cleavage events associated with a protease can both be monitored on the same experimental platform. These efforts will be combined with the development of strategies for probing the kinetics of a binding event on the arrays so that new mechanistic insights into the rate determine step of the binding/cleavage process can be obtained. Accomplishing these aims will combine to illustrate how new synthetic methodology can expand the utility of microelectrode arrays and the types of problems to which they can be applied.